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. 2019 Oct 29;9:15517. doi: 10.1038/s41598-019-51825-2

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Actin binding activity of Y265E ACTN4 is partially regulated by amino terminus phosphomimetic mutations. (A) Immunoblotting of immunoprecipitated WT ACTN4-eGFP and ACTN4Y265E-eGFP using indicated antibodies. (B) Immunoblotting of GFP, actin and GAPDH in total cellular lysate, fraction of cytoplasm and cytoskeleton. (C) Staining of NR6WT cells transiently transfected with Y265E-eGFP or Y4/31/265E-eGFP. Red: Rhodamine phalloidin; Green: fluorescence of ACTN4-eGFP; Blue: DAPI. Scale bar, 20 μm. (D) Cell migration speed of melanoma WM1158 cells transiently expressing WT or indicated eGFP-tagged mutants tracked using live microscopy and analyzed using MetaMorph software. The migratory units stand for the average distance of cells moved within 10 minutes. Cell number (N) = 50. Images are a representative of three independent experiments. Data are mean of ±SD of three independent experiments. Statistical analysis was performed using Student’s t-test. ***p < 0.001.